Effects of adeno‐associated virus‐2‐mediated human BMP‐7 gene transfection on the phenotype of nucleus pulposus cells

Abstract

Bone morphogenetic protein-7 (BMP-7) was found to stimulate the synthesis of proteoglycans (PGs) and collagen type II. To increase the biological function of the nucleus pulposus (NP) cells, the Ad-hBMP-7 vector was also successfully constructed and transfected NP cells. However, the disadvantages of adenovirus limit the usefulness of the Ad-hBMP7 vector for clinical application. The rAAV2 vector has empirical advantages, especially for clinical use, to transfer exogenous genes into cells. The purpose of this study was to first determine whether a rAAV2-hBMP-7 vector could be used to transfect canine NP cells and effect on the biological functions of canine NP cells. The canine NP cells transfected by the rAAV-BMP7 were assessed semi-quaiitatively for BMP-7 expression with real-time PCR and westernbloting. Aggrecan and collagens type I and II secreted by the NP cells were qualitatively assessed at 4, 7, and 14 days post-transfection in the transfection and control groups. We found that rAAV2 can successfully transfer the hBMP-7 gene into canine NP cells. NP cells transfected by the rAAV-hBMP-7 vector express hBMP-7 for at least 14 days. At 7 and 14 days, the expressed hBMP-7 promotes a remarkable and significant accumulation of both proteoglycans (42% and 77% higher than non-transfected cells) (p<0.05) and collagen type II (63% and 94% higher than non-transfected cells) (p<0.05). Thus, we could speculate that the rAAV-based gene delivery technique promotes the expression of proteoglycans and collagen type II of nucleus pulposus cells. Moreover, this technique may be applicable for the future treatment of degenerative disc disease. ß

Human bone morphogenetic protein 7 transfected nucleus pulposus cells delay the degeneration of intervertebral disc in dogs

“…The hBMP7 gene that was transduced into the NP cells by AAV2 could effectively stimulate the synthesis of PGs and collagen II, which were very important in maintaining the tissue structure and the physiological function of the IVD, as previously reported in our in vitro research [19]. Therefore, the advantage of these energetic seed cells for cell transplantation was not only in supplying the quantity of IVD cells in the degenerative disc to produce ECM intended to re-establish healthy disc function, but also in providing cell factors and signaling cues that ameliorate the unbalanced metabolism of the degenerative disc.…”

“…Group A, L1-2, was injected with 0.9% saline (50μL); group B, L3-4, was transplanted with rAAV2-EGFP-NPCs(50μL, 2×10 5 cells); group C, L5-6,was transplanted with rAAV2-hBMP7-NPCs(50μL, 2×10 5 cells). The number of cells injected was chosen according to our previously study and Kenji's study [19,20,22].…”

“…We have already published our in vitro and in vivo work showing that canine NPCs transfected with the recombinant adeno-associated virus-2-mediated human BMP7 (rAAV2-hBMP7) vector could expressed the human BMP7 protein, and could survived in the allogenic transplanted IVD and produced a large amount of PGs and collagen type II, which was considered to have a potential role in preventing the degeneration of IVD allograft [19,20].…”

“…The transfection method and the optimal MOI of 1×10 5 was chosen according to our previous study demonstrating that this MOI produced the highest level of EGFP transgene expression [19].…”

142 Intradiscal Injection of Simvastatin Results in Radiologic, Histologic, and Genetic Evidence of Disc Regeneration in a Rat Model of Degenerative Disc Disease

“…To date, BMP-based treatment in animal and human models of DDD has involved either direct administration of BMP or up-regulation of BMP via alternate routes (such as with adenoviruses) [21][22][23][24][25][26][27][28][29][30][31][32]. However, endogenous up-regulation of BMP may be more effective and less toxic than supraphysiologic exogenous administration.…”